Three phages fS1, fS2and fS3
which utilized Streptomycesdiastaticus,
S.griseus and S.hygroscopicus respectively, as
propagation hosts, were isolated from the soils of jarrah
forest in Western Australia. They were partially characterized
through their physiochemical properties, plaque morphology,
host range and particle morphology. Host range of these three
Siphoviridae (B1) morphotype phages demonstrated a wide
activity spectra within the genus Streptomyces.
Adsorption rate constants and burst sizes of the three phages
were in the range of 1.58x10-7, 1.26x10-7 and 5.97x10-9 ml/min
and 15.5, 22.5, 12.9 virions/cell, respectively. Agarose
electrophoresis of restriction endonuclease digests of the
phage DNAs indicated that the three phages were different.

Diaz et al. (1989) indicated that the isolation and
identification of Streptomyces phages are of interest
for a variety of reasons, which include (i) the problems they
cause to fermentation industries (Chater, 1986), (ii) their
value for typing streptomycetes in taxonomic studies
(Wellington and Williams, 1981; Prauser, 1984; Korn-Wendish
and Schneider, 1992), (iii) their use for the detection and
understanding of host controlled restriction-modification
systems (Diaz et al., 1989), (iv) their utilization as
tools for genetic exchange and analysis in Streptomyces
(Herron and Wellington, 1990), (v) the study of their
general and molecular biology (Lomovskaya et al., 1980)
and ecology (Williams et al., 1987) and most recently
(vi) their use to reduce the numbers of streptomycetes on
isolation plates to select rare actinomycetes (Kurtbke et
al., 1992).

In an attempt to isolate actinoplanetes from jarrah forest
soils of Western Australia, we used three polyvalent
streptomycete phages to reduce the numbers of streptomycetes
on isolation plates to select the targeted taxon. This paper
deals with the partial characterization of these phages and
describes their host range and physiochemical properties.

MATERIALS AND METHODS

Soil samples. Fresh soil samples were collected
from the A horizon of the northern jarrah (Eucalyptus
marginata Donn ex Sm.) forest (near Dwellingup) in Western
Australia. These samples were mixed to form a bulk sample with
a final pH of 6.5 and this heterogeneous mixture was used to
isolate phages.

Phage isolation and purification. Flasks (250ml)
containing 20ml of sterile peptone-yeast extract calcium
(PYCa) broth (Bradley et al., 1961) were inoculated
with 1ml of the spore suspension of the prospective host
streptomycete and 2g of the bulk soil sample. These were then
incubated in a gyrotory shaker (Model G76, New Brunswick
Scientific-Edison, N.J., USA) at 200rpm for 2dd at 28 C. After
incubation, the suspensions from each flask were centrifuged
for 1hr at 2,000g and the supernatants filtered through
sterile Millipore membranes of pore size 0.22æm (Millipore
Corp.) and collected in sterile tubes. Filtrates (0.2ml) were
spotted onto plates of PYCa agar (Bradley et al., 1961)
on which 0.3ml of glycerol suspension of the prospective host
had been spread onto PYCa plates and dried for 30min in a
laminar flow (Vickers and Williams, 1987). The plates were
then incubated at 28 C for 2dd and examined for plaques
(Williams et al., 1980). Single plaques were removed
from the agar plates and resuspended in 1 ml of PYCa broth for
36hrs (Williams et al., 1980). A sample of this broth
was then filtered and spotted on the PYCa plates previously
inoculated with the prospective hosts. Single plaques which
developed on these plates were removed, resuspended, filtered
and these purified phage suspensions were stored at 4 C
(Williams et al., 1980).

Host range. Type strains of Streptomyces
spp. used were obtained from Dr.E. Lacey, McMaster
Laboratory, C.S.I.R.O., Sydney (Table 1). The host range of
the phages were studied by spotting 0.2ml of phage suspensions
containing 107 pfu/ml onto PYCa agar plates each previously
seeded with a glycerol suspension (x106 cfu/ml) of one of the
type strains (Table 2). Phage suspensions were prepared by
ten-fold dilutions of the clear spots on the propagation host.
Clear spot dilution (CSD) is equivalent to the routine test
dilution (RTD) (Wellington and Williams, 1981). The plates
were then incubated for 2dd at 28 C and examined for lysis.

Electron microscopy. A drop of each phage
suspension (107 pfu/ml) was placed on 200-mesh copper grids
with carbon-coated Formvar films and the excess drawn off with
filter paper. A saturated solution of uranyl acetate was then
placed on the grids and the excess drawn off as before.
Specimens were observed with a JEOL-2000 FX II transmission
electron microscope operated at 80 kV.

Effects of physical and chemical agents on phage
propagation and viability. Three different media: peptone-
yeast extract (Bradley et al., 1961), Nutrient agar
(NA) (Difco) and Tryptic soy agar (TSA) (Difco) supplemented
with and without Ca(NO3)2 (0.05%) and with two different
concentrations of NaCl (0.1M, 0.01M) (Brownell et al.,
1967) were tested to determine the effects of these complex
media on phage propagation. The effects of physical and
chemical agents on the phage and the effects of the size of
phage inoculum and host age on the number of plaque forming
units were tested according to the methods described by
Brownell et al. (1967).

Adsorption and one-step growth experiment. The
adsorption rates of the three phages were determined by
measuring residual plaque-forming ability in membrane-filtered
samples of an attachment mixture (Dowding, 1973) and the
adsorption rate constant K ml/min was calculated (Sykes et
al., 1981). A one-step growth experiment was conducted as
described by Dowding (1973).

Restriction endonuclease digestion and agarose gel
electrophoresis. The nucleic acids of the three phages
were isolated according to the methods described by Rodriguez
and Tait (1983). The isolated nucleic acids and lDNA were
digested with the enzymes Hind III, Bam HI
(Gibco BRL) and Eco RI (Progen Industries Ltd.) at 37 C
for 3hrs (Rodriguez and Tait, 1983). Reaction buffers were
supplied with the enzymes. The intact and enzyme treated
nucleic acids were electrophoresed in 1% agarose (Progen
Industries Ltd.) in 40 mM Tris-acetate, pH 8.0 and 2mM EDTA.
Electrophoresis was run on a horizontal apparatus at 8V/cm for
2hrs at room temperature (Rodriguez and Tait, 1983). After
electrophoresis, the gel was stained with ethidium bromide
(0.5æg/ml) and photographed. lDNA was used as size marker
during the examination of the restriction endonuclease
digested phage DNAs.

Electron microscopy. Negatively stained particles
of the three phages fS1, fS2 and fS3
belonged to the Siphoviridae (B1) morphotype (Francki
et al., 1991) and the phages fS1, fS2 had
icosahedral heads 41.2x41.2 and 41.2x41.2nm in diameter
respectively. However, fS3 had an ovoid head
(47.0x32.3nm). The tails of the phages fS1, fS2
and fS3 were 7.0x 159, 7.0x188 and 5.9x253nm
respectively (Figs. 1, A, B and C).

Host range. Phages fS1, fS2 and
fS3 were not species specific and lysed a wide range of
Streptomyces species. However, they did not utilize
Streptoverticillium species (Table 1).

Effect of physical and chemical agents on phage
propagation and viability.
PYCa supplemented with 0.05% Ca(NO3)2 was the best medium for
phage propagation followed by NA and TSA (Table 2). Increased
NaCl concentrations reduced the numbers of congruent plaques
on all three media and no plaque forming units were counted on
TSA when it was supplemented with 0.1M NaCl (Table 2).

The three phages were sensitive to chloroform, thymol,
hydrogen peroxide and ethanol, all of which greatly reduced
the numbers of plaque forming units (Table 2). Freezing at -4
C for 2hrs had an adverse effect on phage titre in particular
on phage fS2. At 4 C phage numbers decreased markedly
with time. Less phages were obtained when propagation hosts
were incubated for 0-5hrs, in comparison to those incubated
for 10-12hrs (Table 3). Greater levels of host inoculum
resulted in greater phage titres and similarly greater phage
inoculum size resulted in greater phage output (Table 3).

Adsorption rate constant, latent period and burst size
of the phages. Adsorption rates of the three phages rising
from spores were 1.58x10^-7, 1.26x10^-7 and 5.97x10^-9 ml/min
respectively. The percentage decrease observed in free phage
numbers was linear between 0 and 20 min. After 20min the
adsorption rates appeared to fall. Counts from samples taken
after 35-40min suggested that infected cells were lysing and
liberating phage. Latent period values, obtained for the three
phages, were 35, 40 and 40min, which confirmed the
observations made during the adsorption experiments.

The rise periods of the phages were 40, 30 and 40min
respectively, with average burst sizes of 15.5, 22.5
and 12.9 virions/cell. The second burst began at around
70-80min, nearly 30-40min after the first burst began,
which confirmed the minimum latent period of 30-40min for the
three phages (Table 4).

Restriction endonuclease digestion of phage DNAs.
The phages f S1, fS2 and fS3 were
readily digested by Bam HI but not the other enzymes
used (Fig. 2). The three phages did not share similar restriction
fragments indicating that they were different. This was
supported by particle morphology and the results
obtained in growth kinetics experiments (Table 4).

DISCUSSION

Phages active against Streptomyces spp., the
predominant actinomycete genus in soil, are readily detected
and it appears that streptomycete phages are widespread in the
soil environment (Williams and Lanning, 1984).

Table 3. Effects of age and inoculum size of
propagation hosts on phage production and effects of phage
inoculum size on the yield of phages. Propagation hosts used
for phages fS1, fS2 and fS3 were Streptomyces diastaticus
(ATCC 3315),S.griseus (NCTC 7807) and
S.hygroscopicus (ATCC 31955) respectively.

The rapid isolation of Streptomyces phages without
any enrichment techniques agrees with these previous findings.
The isolated phages are of the same morphological type as
other streptomycete phages (Ackermann et al., 1985).

The broad activity spectra are in agreement with the
previous findings that common phage susceptibility is one of the
distinctive characters of the members of the genus
Streptomyces (Wellington and Williams, 1981; Prauser, 1984).

The increase in the pfu of phages, when Ca(NO3)2 was added
into the media used in this study, confirmed the findings of
Adams (1959) who concluded that divalent cations such as Ca++
are required for adsorption of phages to the receptors. Gold
(1959) also noted that addition of divalent cations increased
the size and number of plaques produced by phages. The increased
NaCl concentrations reduced the numbers of congruent
plaques on all three media types and no plaque forming units
were counted on TSA when it was supplemented with 0.1M NaCl.
These findings agree with Walton (1951) who concluded that
certain concentrations of alkaline cations, sodium, potassium
and ammonium completely inhibit multiplication of actinophages.
The physical and chemical treatments used had adverse effects
on all three phages. The phage fS2 isolated to
Streptomycesgriseus (NCTC 7807) was
the most affected. Although most phages are resistant to
detergents and other chemical and physical agents, their
inactivation by agents such as chloroform is commonly observed
(Goyal, 1987).

The burst sizes obtained for the phages were lower than
the ones obtained by Dowding (1973) but higher than some of
the values obtained by Sykes et al. (1981) for phages
that utilized neutrophilic hosts. However, as observed by
Jones and Bradley (1965), many factors can influence phage
burst size, such as the filamentous nature of the host, spore
clumping and host age. Dowding (1973) also concluded that
actinomycetes, including representatives of the genus
Streptomyces, do not germinate synchronously, with the
result that by the time a high enough proportion of spores
have germinated, some of them will have formed large clumps of
mycelium. These clumps can adsorb a great many phage particles
yet continue to plate out as single colony-forming units.
Therefore, the concept of multiplicity of infection will
remain meaningless in actinomycete host-phage systems until a
method is found to achieve synchronous and uniform spore
germination.

This is the first record of the detection of Streptomyces
phages from the jarrah forest soils of Western Australia. As
indicated by Williams and Lanning (1984) phages are a neglected
ecological entity. Isolation of phages from different substrates
in neglected environments will increase our knowledge on the
ecology of species, genus and family specific phages and provide
additional information on phage ecology and phage-host interactions
(Williams and Lanning, 1984; Kurtbke and Williams, 1991).

ACKNOWLEDGEMENTS.

This work was sponsored by Alcoa Australia Ltd. We
thank Dr. I.Colquhoun for his encouragement. Mr.S.Wylie for his
kind help during the restriction endonuclease digestion of
the phage DNAs and Prof. M.J.K.Jones for allowing us to use
his facilities. Mr.S.Parry (University of Western
Australia, Electron Microscopy and Microanalysis Centre) assisted
with the preparation of the electron micrographs.

Herron, P.R. & E.M.H.Wellington (1990). New method
for extraction of streptomycete spores from soil and
application to the study of lysogeny in sterile amended and
nonsterile soil. Appl. Environ.Microbiol., 56:
1406-1412